How do I reverse translate (back translate) a protein sequence?
Create, Import or Open a Protein Sequence
Open or Import a protein sequence.
If the protein sequence does not have a CDS feature then select the protein sequence and click Features → Add Feature and add a feature of type CDS. Give the feature a "Name" and click OK.
In Map view or Sequence view click the CDS feature to select the protein sequence, or in Sequence view, select the region of the protein to be reverse translated.
Click Actions → Reverse Translate....
Set the "Genetic Code" to use for reverse translation.
Select the method for reverse translation:
1) Random chosen codons. This method will randomly assign a synonymous codon for each amino acid.
2) Degenerate codons. This method will create a degenerate sequence, and where possible, each codon will encode all possible synonymous codons for a particular amino acid. Note that amino acids with 6 synonymous codons (Leu, Arg and Ser) cannot be represented by a single unambiguous degenerate sequence. For example, Leu can be encoded by CTN or TTR. The combined YTN will also encode Phe. Check the option "Allow ambiguous codons" if required.
3) Use a Codon usage table. Choose to back translate using a codon usage table (CUT)†. Choose whether to use "Preferred Codons" (using most frequent synonymous codon only), or "Codons chosen with probabilities based on the frequency distribution".
Give the new CDS DNA product a name, then click Reverse Translate.
† For increased likelihood of good recombinant protein expression it is important you use a codon usage table that specifies codons frequencies observed for highly expressed host proteins in your expression host. We recommend you consult the literature for advice on the recommended codon usage frequencies for your expression host.
View the New DNA Sequence
The new "reverse translated" sequence will be annotated with a translated CDS Feature. Double click the feature and rename it if desired.
To view the relative codon frequencies for the new sequence, select the CDS feature then click Features → Codons → Show Codon Frequencies.
View the codon frequencies for the new CDS.
To view the codon usage table used for the reverse translation process, click Tools → Show Codon Usage tables, then click "Codon Usage Table" to select the codon usage table used for back translation.
The relative frequencies of synonymous codons observed for the new CDS and the CUT used to generate the new sequence should be roughly the similar.
Replace Rare Codons (Optional)
If the CUT used to optimize a CDS contains codons with relatively low frequencies then you can replace those codons with preferred codons. Select the CDS, then click Features → Codons → Choose alternative Codons.
Set the "Codon Usage Table" to use, set the "rare" codon threshold, any "rare" codons in the sequence will be highlighted yellow.
then choose Replace Codons → Replace Rare Codons. All rare codons will be replaced with "preferred" codons and highlighted blue.
If you make a mistake then click Edit → Undo to undo changes and start again.
Click OK to close the window and accept the changes to the sequence.
View the Codons used by the Reverse Translated and Optimized DNA Sequence
To view the relative codon frequencies for the new sequence, select the CDS feature then click Features → Codons → Show Codon Frequencies.
In this example, all codons that occur with a frequency of less than 10% in the CUT will be considered rare (highlighted yellow above), and if present in your CDS will replaced with a preferred codon and so have a frequency of zero (0).
Save the DNA sequence
Click File → Save to save the new DNA sequence.